Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R_cryst of 0.199 (R_free = 0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving α1-α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å², which is considerably higher than the 660 Å² reported for the class I major histocompatibility complex structures.     

Residue-Specific Analysis of Frustration in the Folding Landscape of Repeat β/α Protein Apoflavodoxin

Flavodoxin adopts the common repeat β/α topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e., at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, ?-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding, while proper alignment on both sides of β-strand 3 is necessary for productive folding.     

Binding Modes of Thioflavin-T to the Single-Layer β-Sheet of the Peptide Self-Assembly Mimics

Although the amyloid dye thioflavin-T (ThT) is among the most widely used tools in the study of amyloid fibrils, the mechanism by which ThT binds to fibrils and other β-rich peptide self-assemblies remains elusive. The development of the water-soluble peptide self-assembly mimic (PSAM) system has provided a set of ideal model proteins for experimentally exploring the properties and minimal dye-binding requirements of amyloid fibrils. PSAMs consist of a single-layer β-sheet (SLB) capped by two globular domains, which capture the flat, extended β-sheet features common among fibril-like surfaces. Recently, a PSAM that binds to ThT with amyloid-like affinity (low micromolar K_d) has been designed, and its crystal structure in the absence of bound ThT was determined. This PSAM thus provides a unique opportunity to examine the interactions of ThT with a β-rich structure. Here, we present molecular dynamics simulations of the binding of ThT to this PSAM β-sheet. We show that the primary binding site for ThT is along a shallow groove formed by adjacent Tyr and Leu residues on the β-sheet surface. These simulations provide an atomic-scale rationale for this PSAM's experimentally determined dye-binding properties. Together, our results suggest that an aromatic-hydrophobic groove spanning across four consecutive β-strands represents a minimal ThT binding site on amyloid fibrils. Grooves formed by aromatic-hydrophobic residues on amyloid fibril surfaces may therefore offer a generic mode of recognition for amyloid dyes.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H_f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.     

Improving the resistance of a eukaryotic β-barrel protein to thermal and chemical perturbations

β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the “mitochondrial porin family” and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for β-barrel membrane proteins, we have identified three unfavorable β-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.     

Chemical shift assignments of the canecystatin-1 from Saccharum officinarum

Cystatins are cysteine proteases inhibitors that are widely distributed among insects, mammalians and plants. Here we report the complete resonance assignment of canecystatin-1 from Saccharum officinarum obtained by heteronuclear multidimensional high-resolution nuclear magnetic resonance spectroscopy. The consensus chemical shift index was calculated and showed the presence of one α-helix (residues 27–43) and three β-strands (residues 48–74, 78–89 and 94–104), a secondary structure pattern that suggests a domain-swapped structure as presented by stefin B and human cystatin C, opposed to the monomeric structure yet found in other phytocystatins like oryza and pineapple cystatin.     

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 Å. The protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.     

A model of the complex between human β-microseminoprotein and CRISP-3 based on NMR data

β-Microseminoprotein (MSP), a 10 kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. ¹⁵N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising β-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that β-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.     

Crystal structure of the peptidoglycan recognition protein at 1.8 A resolution reveals dual strategy to combat infection through two independent functional homodimers

The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a = 87.0 Å, b = 101.7 Å and c = 162.3 Å having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 Å resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central β-sheet with five β-strands, four parallel and one antiparallel, and three α-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix α2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Model for Substrate Interactions in C5a Peptidase from Streptococcus pyogenes: A 1.9 Å Crystal Structure of the Active Form of ScpA

The crystal structure of an active form of ScpA has been solved to 1.9 Å resolution. ScpA is a multidomain cell-envelope subtilase from Streptococcus pyogenes that cleaves complement component C5a. The catalytic triad of ScpA is geometrically consistent with other subtilases, clearly demonstrating that the additional activation mechanism proposed for the Streptococcus agalactiae homologue (ScpB) is not required for ScpA. The ScpA structure revealed that access to the catalytic site is restricted by variable regions in the catalytic domain (vr7, vr9, and vr11) and by the presence of the inserted protease-associated (PA) domain and the second fibronectin type III domains (Fn2). Modeling of the ScpA-C5a complex indicates that the substrate binds with carboxyl-terminal residues (65-74) extended through the active site and core residues (1-64) forming exosite-type interactions with the Fn2 domain. This is reminiscent of the two-site mechanism proposed for C5a binding to its receptor. In the nonprime region of the active site, interactions with the substrate backbone are predicted to be more similar to those observed in kexins, involving a single β-strand in the peptidase. However, in contrast to kexins, there would be diminished emphasis on side-chain interactions, with little charged character in the S3-S1 and S6-S4 subsites occupied by the side chains of residues in vr7 and vr9. Substrate binding is anticipated to be dominated by ionic interactions in two distinct regions of ScpA. On the prime side of the active site, salt bridges are predicted between P1′, P2′, and P7′ residues, and residues in the catalytic and PA domains. Remote to the active site, a larger number of ionic interactions between residues in the C5a core and the Fn2 domain are observed in the model. Thus, both PA and Fn2 domains are expected to play significant roles in substrate recognition.     

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li⁺-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr⁹⁰ is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase (FBPase,² EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2). They possess a common structural core with the active site lying between α+β and α/β domains (3). Li⁺-sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulin-dependent diabetes (5, 6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7, 8).Presently, five different classes of FBPases have been proposed based on their amino acid sequences (FBPases I to V) (9–11). Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in various prokaryotes. Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11). Many organisms have more than one FBPase, mostly the combination of types I + II or II + III, but no bacterial genome has a combination of types I and III FBPases (9). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Escherichia coli, most bacteria, a few archaea, and all eukaryotes (9, 11–15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9, 16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10, 21).Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10, 11, 19, 20, 22, 23). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved α-helices and β-sheets (α-β-α-β-α) (2, 19, 24). ST0318 (an FBPase V enzyme) is composed of one domain with a completely different four-layer α-β-β-α fold (10). The FBPases from these three classes (I, IV, and V) require divalent cations for activity (Mg²⁺, Mn²⁺, or Zn²⁺), and their structures have revealed the presence of three or four metal ions in the active site.E. coli has five Li⁺-sensitive phosphatases as follows: CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a 3′-phosphoadenosine 5′-phosphatase involved in the cysteine biosynthesis pathway (25, 26), whereas SuhB is an inositol monophosphatase (IMPase) that is also known as a suppressor of temperature-sensitive growth phenotypes in E. coli (27, 28). Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12). This enzyme has been characterized both biochemically and structurally and shown to be inhibited by low concentrations of AMP (IC₅₀ 15 μm) (11, 29, 30). The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn²⁺-dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp^- phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).In this study, we present the first structure of a class II FBPase, the E. coli GlpX, in a free state and in the complex with FBP + metals or phosphate. We have demonstrated that the fold of GlpX is similar to that of the lithium-sensitive phosphatases. We have identified the GlpX residues important for activity and proposed a catalytic mechanism. We have also showed that YggF is a third FBPase in E. coli, which has distinct catalytic properties and is more sensitive than GlpX to the inhibition by lithium or phosphate.